Apparatus for maintaining a controlled environment

ABSTRACT

A lyophilization nest and method of using the same is described herein. In various embodiments, the lyophilization nest is configured to support one or more receptacles each supporting one or more substances within an interior space of the lyophilization nest. The interior space may be in fluid communication with the exterior of the lyophilization nest through one or more vent holes extending through a surface of the lyophilization nest. Each of the one or more vent holes have a corresponding sealing element configured to selectively form an air-tight seal within the vent holes, such that a controlled environment may be maintained within the interior space when the ambient conditions surrounding the lyophilization nest are not lyophilization conditions. The one or more sealing elements may be operable while the lyophilization nest is positioned within a sealed lyophilizer by depressing the sealing elements into corresponding vent holes to form the air-tight seal.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation application claiming the benefit under 35 U.S.C.§ 120 of the filing date of non-provisional patent application Ser. No.16/560,476 filed Sep. 4, 2019, which is a continuation application ofnon-provisional patent application Ser. No. 15/745,834 filed Jan. 18,2018, now U.S. Pat. No. 10,443,935, issued Oct. 15, 2019, which is a 35U.S.C. 371 National Phase Entry Application from PCT/US2016/045166,filed Aug. 2, 2016, which claims the benefit under 35 U.S.C. § 119(a) ofthe filing date of U.S. Provisional Application No. 62/200,370, filedAug. 3, 2015, the respective disclosures which are incorporated hereinby reference.

BACKGROUND

Lyophilization is a process used to remove water from substancesallowing these substances to be stored for longer periods of timewithout material deterioration. Typically these substances are ofbiological or synthetic origin and may include antibiotics,pharmaceuticals, chemicals, sera, vaccines, cells, tissues, proteinand/or nucleic acids.

Substances can be lyophilized by placing individual vials or amulti-well plate containing one or more substances into a lyophilizer,which generates and maintains a controlled environment therein duringthe lyophilization process. The environmental factors that arecontrolled by the lyophilizer include, but are not limited to,temperature, air pressure/vacuum level, humidity, and/or gas content.After lyophilization is complete, the seal on the lyophilizer isreleased, and consequently the controlled environment within thelyophilizer is diluted with the air surrounding the lyophilizer, whichcan be detrimental to lyophilized substances. A primary consequence ofcontacting lyophilized substances with the air from outside of thelyophilizer is that the air generally contains moisture (e.g., watervapor) which may rehydrate (at least partially) the lyophilizedsubstances. Depending on factors such as the time of year, the roomair-conditioning, the number of people in a room and the exposure time,lyophilized substances in contact with air from outside of thelyophilizer can undergo from 40% to 70% rehydration. Introducingmoisture to the lyophilized substances can negatively affect thestability of the substances. If the substances are not going to be usedimmediately, the vials and/or wells of the multi-well plates can besealed by placing a foil or other sealing material over the wells and/orvials, thereby sealing and isolating the lyophilized substances from theair. The seal provides a barrier between environmental conditionssurrounding the plate and the substances located within each vial orwell of the plate after the plate has been removed from the lyophilizer.Thus, the seal impedes rehydration of substances that results fromabsorbing water vapor from the atmosphere. The sealed vials and/orplates containing the one or more substances can then be stored or soldin commerce for later use. The seals can then be broken such that thestored substances within one or more of the vials and/or individualwells may be accessed during use.

The process of sealing receptacles after removal from the lyophilizerexposes the individual substances to unfavorable environmentalconditions during the period of time between removal of the receptaclefrom the lyophilizer until the receptacle is sealed. While this exposuretime can vary depending on the number of vials and/or wells of themulti-well plate to be sealed, the number of technicians available toseal the vials and/or wells, the transportation distance between thelyophilizer and the sealing station, and other factors experiencedduring a lyophilization procedure, the exposure time for lyophilizedsubstances in a high-production facility may be between 4-5 hours. As aresult, the lyophilized substances in each vial or well of themulti-well plate may be exposed to uncontrolled and potentiallyunfavorable environmental conditions, including moisture, prior to beingsealed.

Prolonged exposure of the lyophilized substances to these uncontrolledenvironmental conditions during the period of time between removal ofreceptacles from the lyophilizer until the receptacles are sealed may bereduced by placing the receptacles into a glovebox (e.g., the Purelab HE4 GB 2500 Glovebox, available from Innovating Technology, Inc.Newburyport, Mass.) immediately after removal from the lyophilizerwithin which an operator may seal each of the receptacles. However,manipulation of the receptacles may be cumbersome when using a glovebox. Additionally, the moisture vapor exposure time during transfer ofthe receptacles from the lyophilizer to the glovebox may still besignificant and may lead to moisture absorption by the lyophilizedsubstances. Furthermore, the environment within a glovebox cannot bemade the same as the environment within the lyophilizer itself, and sothe lyophilized substances remains exposed to an unfavorable humiditylevel for a long period of time before being sealed.

A lyophilizer may be operated within a dry room with low humidity. Onremoval of receptacles from the lyophilizer, substances are exposed onlyto the dry room prior to sealing. However, dry rooms require precisionclimate control systems for maintaining low humidity within aroom-volume sized to accommodate at least one operator. The humiditylevels are again not the same as those within the lyophilizer, though,and the mere presence of an operator within the dry room furthernegatively impacts the environmental conditions. Dry rooms are expensiveto maintain, subject to contamination, and cumbersome to operate becauseconditions may be hazardous for individuals working inside of theserooms, and accordingly the amount of time an individual may work withinthese rooms may be limited.

Large stoppered individual vials storing a single substance can beconfigured for the stoppers to engage while the individual vials arelocated within a sealed lyophilizer. However, such individual vials aredifficult to manage, bulky to store, and not usually compatible withlaboratory equipment designed for handling samples in a multi-wellformat.

BRIEF SUMMARY

Described herein a lyophilization nest for preparing lyophilizedsubstances, comprising: a base comprising a bottom plate having a baseedge extending upwardly from a perimeter thereof, the bottom platehaving a top surface adapted to support a single receptacle or aplurality of receptacles thereon; a cover comprising a top plate havinga cover edge extending downwardly from a perimeter thereof, the topplate having one or more vent holes extending therethrough; an interiorspace defined by the base and the cover when the base and the cover arein the closed relationship, a gasket situated between the base edge andthe cover edge and forming an air tight seal between the base edge andthe cover edge when compressed; one or more sealing elements, each ofthe sealing elements being in closeable engagement (e.g., slidingengagement, hinging engagement, swinging engagement, rotary engagement,and the like) with a corresponding one of the vent holes, such that thesealing elements and the corresponding vent holes are operable between:(i) an open configuration in which the positions of the sealing elementsrelative to the corresponding vent holes permit fluid communicationbetween the interior space and the air outside of the lyophilizationnest when the base and the cover are in the closed relationship; and(ii) a closed configuration in which the positions of the sealingelements relative to the corresponding vent holes do not permit fluidcommunication between the interior space and the air outside of thelyophilization nest when the base and the cover are in the closedrelationship, such that the interior space is sealed off to air externalthe lyophilization nest when the base and the cover are in the closedrelationship.

In various embodiments, each of the one or more sealing elements is aflexible plug that comprises a sealing cap and a body portion dependingtherefrom, the body portion being in a sliding closeable engagement witha corresponding one of the vent holes and having one or more vent slotsformed therein. Each of the sealing elements is configured to bepositioned in a corresponding vent hole, such that (i) the interiorspace is in fluid communication with the air outside of thelyophilization nest via the vent slots when the sealing element and thecorresponding vent hole are in the open configuration; and (ii) theinterior space is not in fluid communication with the air outside of thelyophilization nest when a bottom surface of the sealing cap is insealing contact with a top surface of the top plate when the sealingelement and the corresponding vent hole are in the closed configuration.

In various embodiments, at least one of the base and the cover comprisealuminum. Various embodiments may comprise a hinge along one or moreedges of the top plate or the base to provide a clamshell configurationbetween the base and the cover.

In various embodiments, the base interior is configured to support oneor more receptacles therein. In various embodiments, the receptacles mayeach comprise a plurality of lyophilization wells configured to besealed and a plastic and/or low moisture vapor transmission ratematerial, such as a cyclic olefin copolymer. Moreover, in variousembodiments, the top surface of the bottom plate comprises one or morereceptacle receiving portions formed thereon, each of the receptaclereceiving portions having features that conform to at least a bottom endof the one or more receptacles to be received thereon and areconstructed to conduct heat between the base and the one or morereceptacles supported thereon. In various embodiments, each of the oneor more receptacles to be supported within the lyophilization nestcomprise a plurality of lyophilization wells, and each of the receptaclereceiving portions comprises a plurality of well receiving featuresconforming to the shapes of at least the bottom ends of lyophilizationwells of the corresponding receptacle. Moreover, in various embodiments,the lyophilization nest additionally comprises one or more receptacleframes situated on a top surface of the bottom plate, each receptacleframe configured to support a plurality of receptacles (e.g., fourreceptacles).

In various embodiments the lyophilization nest further comprises one ormore fasteners for securing the cover to the base. The fasteners of thelyophilization nest are configured to compress the gasket between thebase edge and the cover edge to form the air tight seal when the baseand the cover are in the closed relationship. As an example, thefastener further comprises a base engagement member such as a base pin,a cover engagement member such as a cam lever, and a linking member thatlinks the other two members. As further examples, the fastener can be alatch, a bolt, a clamp, or other structure that compresses the gasketbetween the base edge and the cover edge to form the air tight seal whenthe base and the cover are in the closed relationship.

Various embodiments herein are directed to a lyophilization system forlyophilizing one or more substances, the lyophilization systemcomprising: a sealable enclosure defining a chamber and having aplurality of shelves. In various embodiments, the plurality of shelvesare a top shelf and a bottom shelf contained within the chamber. Invarious embodiments, the plurality of shelves are at least one top shelfand at least one bottom shelf. In various embodiments, the plurality ofshelves are a top shelf, a bottom shelf and at least one middle shelfthat can be referred to as a top shelf and/or bottom shelf depending onits spatial relationship to a lyophilization nest in the lyophilizationsystem. In various embodiments, the plurality of shelves are a topshelf, a bottom shelf and at least two middle shelves that can bereferred to as a top shelf and/or bottom shelf depending on theirspatial relationship to a lyophilization nest in the lyophilizationsystem. In various embodiments, the plurality of shelves are at leastone top shelf and at least one bottom shelf, each relative to alyophilization nest in the lyophilization system. In variousembodiments, a lyophilization nest is situated on a bottom shelf. Invarious embodiments, at least one of a top shelf and a bottom shelf iscapable of an automated movement that causes the top shelf to engage thesealing elements, thereby altering the sealing elements and thecorresponding vent holes from the open configuration to the closedconfiguration. In various embodiments, the top shelf is positioned abovethe lyophilization nest and is configured for automated downwardmovement toward a top surface of the cover of the lyophilization nest.

Various embodiments herein are directed to a lyophilization system forlyophilizing one or more substances, the lyophilization systemcomprising: a sealable enclosure defining a chamber and having at leastone top shelf and at least one bottom shelf contained within thechamber; and at least one lyophilization nest situated on a bottomshelf. In various embodiments, at least one of a top shelf and a bottomshelf is capable of an automated movement that causes the top shelf toengage the sealing elements of the lyophilization nest(s) on the shelfbelow (bottom shelf), thereby altering the sealing elements and thecorresponding vent holes from the open configuration to the closedconfiguration. In various embodiments, the top shelf is positioned abovethe lyophilization nest(s) and is configured for automated downwardmovement toward a top surface of the cover of the lyophilizationnest(s). Various embodiments are directed to a method for lyophilizingone or more substances, comprising positioning a lyophilization nest ona bottom shelf contained within a chamber of a lyophilizer, wherein thelyophilization nest is supporting one or more receptacles within aninterior space of the lyophilization nest, and at least one of thereceptacles contains one or more substances to be lyophilized. Theinterior of the closed lyophilization nest is in fluid communicationwith the air outside of the lyophilization nest through one or more ventholes extending through a top plate of a cover of the lyophilizationnest and having a sealing element in sliding engagement therewith. Themethod additionally comprises steps for closing the chamber containingthe lyophilization nest, creating a controlled environment havinglyophilization conditions within the chamber for a period of timesufficient for the substance(s) contained in the receptacles to belyophilized. Moreover, the method may comprise causing at least one of abottom shelf and a top shelf contained within the chamber to move sothat the top shelf engages the sealing elements, thereby closing thevent holes so that the interior space of the lyophilization nest issealed off from the air outside of the lyophilization nest. The sealedlyophilization nest contains within the interior space, theenvironmental conditions from the sealed lyophilization chamber, whichincludes a low moisture content, and may further include other factorsfrom the environment of the sealed chamber such as nitrogen gas. Thesealed lyophilization nest is no longer in fluid communication with thesealed chamber. Thus the chamber can be unsealed, which results in theintroduction of environmental conditions that are typically unfavorableto maintaining the lyophilized substance(s), without exposing thelyophilized substance(s) within the sealed lyophilization nests to theunfavorable environmental conditions.

In various embodiments, creating within a lyophilization chamber acontrolled environment for lyophilization comprises generating a vacuumand cycling between temperatures below freezing, which will dry thesubstance(s) within the chamber. At the end of these steps in thelyophilization process, the humidity level is near zero. In order tounseal the lyophilization chamber, the vacuum must first be released.The vacuum is preferably released by flooding the chamber with nitrogengas. A high nitrogen environment is preferred because of its lowmoisture content. It is preferred that the lyophilization nest is sealedfollowing the introduction of nitrogen into the chamber, thereby sealinginto the interior of the lyophilization nest a high nitrogenenvironment. However, this is not mandatory, and the lyophilization nestcan be sealed any time following drying of the substances within. Oncethe vacuum has been released, the chamber door can be opened.

In various embodiments, the method may additionally comprise accessingand removing the receptacles from the lyophilization nest, and sealingthe receptacles so as to form an air tight seal between each of aplurality of wells of each of the receptacles, thereby isolating theinterior of the wells from environmental conditions that negativelyimpact a lyophilized substance in the well. In various embodiments, thereceptacle is sealed by securing a low moisture vapor transmission film,such as, a laminate structure comprising a layer of aluminum foil to atop surface of each of the receptacles to thereby form the airtight sealbetween the wells and the air exterior the sealed wells.

In various embodiments, the relative humidity within the interior spaceof the lyophilization nest remains at 10%, more preferably at less than10%, more preferably at less than 5%, more preferably at approximately0% relative humidity for at least four hours, or for at least 8 hours,or for an amount of time from four hours to eight hours. In variousembodiments, the absolute humidity within the interior space of thelyophilization nest remains at 2.3 grams of water per cubic meter ofair, more preferably at less than 2.3 grams of water per cubic meter ofair, more preferably at less than 1.15 grams of water per cubic meter ofair, more preferably at less than 0.23 grams of water per cubic meter ofair, more preferably at approximately at 0.0 grams of water per cubicmeter of air for at least four hours, or for at least eight hours, orfor a time period from four hours to eight hours.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Reference will now be made to the accompanying drawings, which are notnecessarily drawn to scale, and wherein:

FIG. 1 shows a lyophilization nest in a sealed configuration accordingto one embodiment;

FIG. 2 shows an exploded view of a lyophilization nest according to oneembodiment;

FIG. 3 shows a bottom perspective view of a lyophilization nest coveraccording to one embodiment;

FIGS. 4A-4C show various positions of a sealing element within a venthole according to one embodiment;

FIG. 5 shows a cross-sectional view of a lyophilization nest having areceptacle positioned therein according to one embodiment and with a topshelf and a bottom shelf in contact with the external surfaces of thecover and the base;

FIGS. 6A-6C show various views of a receptacle according to oneembodiment; and

FIGS. 7-9 include cross-sectional views of a lyophilization nest invarious configurations according to one embodiment, the lyophilizationnest containing receptacle(s) in the interior space of the nest. FIGS.7-9 also illustrate a chamber comprising a top shelf and a bottom shelfshown in various positions external the cover and base of the nest.

DETAILED DESCRIPTION

The lyophilization nest will now be described more fully hereinafterwith reference to the accompanying drawings, in which some, but not allembodiments of the nest are shown. Indeed, the lyophilization nest maybe embodied in many different forms and should not be construed aslimited to the embodiments set forth herein. Rather, these embodimentsare provided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Lyophilization is a well-known process for drying a substance so topreserve that substance. The primary mechanism that allows forlyophilization is sublimation, whereby ice is directly converted towater vapor, without passing through the intermediary stage of a liquid.Rather than through heating, this is done by removal of pressure so thatthe ice boils without melting. The result is a substance whose structureis largely preserved, which has a negligible water content, and whichcan be stored at room temperatures and pressures. In order to lyophilizea substance, a number of factors of the environment surrounding thesubstance are altered compared to the more typical levels for thesefactors in the environment. A number of environmental factors arefrequently altered during a lyophilization reaction, including one ormore of temperature, air pressure, atmospheric gas content, andhumidity. Those skilled in the art of lyophilization will readilydevelop and implement a set of environmental conditions to achievelyophilization of a substance of interest. The term “controlledenvironment” and various forms thereof are used herein in reference tothe an environment wherein a number of the factors are altered in orderto lyophilize a substance or to maintain a low water content in asubstance that has been lyophilized. As used herein, an “uncontrolledenvironment” and related terms refers to an environment with factorsthat are unfavorable to lyophilization of a substance or to maintaininga low water content in a substance that has been lyophilized. It isnoted that uncontrolled environment is not being used herein to indicatethat an environment is not controlled in some manner (e.g., room airconditioning and the like), but rather is being used herein toconveniently refer to environmental conditions that are unfavorabletowards lyophilizing a substance and maintaining low water content inthe substance following lyophilization.

Various embodiments are directed to a lyophilization nest configured tomaintain a controlled environment having at least a low humidity levelaround one or more substances after completion of a lyophilizationprocess using a lyophilization system. The lyophilization system,comprising a lyophilizer, a lyophilization nest, and one or morereceptacles each containing one or more substances, is configured tolyophilize the substance(s) and maintain a controlled environment aroundthe substance(s) after the lyophilization process.

The alteration of an environmental factor is often within rangestypically achievable by commercially available lyophilizers. A number ofthe environmental factors are reduced to a negligible level, such thatfurther reduction would not offer significant additional benefit inlyophilization or preservation of samples. One such factor is humiditywherein it is desirable to bring the relative water vapor content of theair close to zero percent. Humidity can be expressed as a relative valuecompared with maximum saturation under given conditions or an absolutevalue measured in grams of water per cubic meter. A 1% relative humidityat 25° C. is equivalent to an absolute humidity of 0.23 g/m³ and arelative humidity of 5% at 25° C. is equivalent to 1.15 g/m³.

For example, relative humidity within a lyophilizer may be reduced toapproximately 0% (undetectable with conventional equipment) or less than1%. Absolute humidity can be reduced to <0.23 g/m³. A controlledenvironment can also include altering the amount of an inert gas, suchas nitrogen, to a greater or lesser proportion than present in theambient atmosphere. A controlled environment can thus be characterizedby any or all of reduced atmospheric pressure (vacuum), reducedatmospheric water vapor content (humidity), increased inert gas content,or reduced atmospheric temperature; compared to the surroundinguncontrolled environment. Measurements for these altered factors aregenerally known, formulae for converting between relative and absolutevalues thereof are readily achieved. Various embodiments of theapparatus and methods of its use described herein will maintain acontrolled environment around one or more lyophilized substances afterthe controlled environment within a lyophilizer has been replaced withan atmosphere comprising levels of factors that are less desirable forthe lyophilized substance(s) than is the controlled environment.

The lyophilization nest is based in part on the insight thatconventional formats for lyophilizing and then resealing substance(s) inreceptacles (e.g., vials and/or multi-well plates) may result inexcessive rehydration of the lyophilized substance(s) due to prolongedcontact with a surrounding uncontrolled environment from the time ofreleasing the seal of a lyophilizer until the individual wells areresealed. Excessive hydration can reduce the activity or shelf-life ofsubstance(s), or in some cases can even result in an exothermic andpotentially dangerous reaction. This excessive contact may result from awork flow that requires the resealing process be performed at a workstation located some distance away from the lyophilizer, or at latertime after the release of the seal of the lyophilizer. Accordingly,herein described is an apparatus for reducing, minimizing and/oravoiding potentially detrimental exposure of lyophilized substance(s) toundesirable levels of environmental factors in an uncontrolledenvironment, and thereby absorbing moisture. The apparatus can be usedwith or without conventional means of reducing rehydration of samples,such as glove boxes and dry rooms.

Exposure of lyophilized substances to undesirable levels ofenvironmental factors in an uncontrolled environment may be avoided byutilizing a sealable lyophilization nest apparatus that preserves withinthe apparatus a controlled environment formed in the chamber of a sealedlyophilizer. The apparatus comprises in its interior one or morereceptacles containing in a receptacle well one or more substances forlyophilization. The interior of the apparatus is in fluid communicationwith the controlled environment formed within the lyophilizer chamberduring lyophilization and for a period of time sufficient to lyophilizethe substance(s). Following lyophilization, fluid communication betweenthe lyophilization chamber and the interior of the apparatus is thenprevented by sealing the apparatus, thereby isolating the apparatusinterior away from air in the chamber. The controlled environment isthen altered within the chamber unsealing the chamber and allowing anuncontrolled environment inside the chamber. Though, because theinterior of the apparatus is sealed, the environment in the interior ofthe apparatus remains favorable to maintaining the lyophilizedsubstance(s) therein. It is noted that all of the environmental factorswithin the apparatus are not necessarily at the same levels as those ofthe controlled environment formed in the lyophilization chamber duringlyophilization. For example, the temperature of the apparatus interiormay change from that used during lyophilization. But, the environmentalfactors within the sealed apparatus interior are favorable formaintaining the lyophilized substance, such that rehydration of thelyophilized substance is restricted. The apparatus, when not in fluidcommunication with the environment outside of the apparatus, canmaintain a favorable environment around the receptacle(s) and within thewells after release of the seal of the lyophilizer chamber until anoperator is prepared for accessing the individual receptacle(s) orwell(s) within such a receptacle (e.g., the individual wells).

A receptacle is a discrete and contiguous vessel for containing one ormore substances. A receptacle can have one or more wells, each well forcontaining a substance. A receptacle with one well is referred to asingle-well receptacle. A receptacle with multiple wells, such as amulti-well plate is referred to as a multi-well receptacle.

In various embodiments, the apparatus may be sealable and portable, suchthat the apparatus containing the receptacle(s) may be removed from thechamber of a lyophilizer and relocated to the appropriate work station(e.g., a clean room, a countertop, or a dry room) for sealing ofindividual receptacle(s) or individual wells therein. The apparatus isreferred to herein as a lyophilization nest because it provides atemporary storage unit for any lyophilized substances therein during theperiod of time between the release of the lyophilizer seal and thebeginning of the resealing process for receptacles containing thelyophilized substance(s).

In brief, in various embodiments substance(s) to be lyophilized areplaced in one or more wells of a receptacle(s). The receptacle(s) areplaced within a lyophilization nest configured to support thereceptacle(s) within an interior space of the lyophilization nest. Theinterior space of the lyophilization nest may be in fluid communicationwith the exterior of the lyophilization nest through one or more ventholes extending through a surface of the lyophilization nest (e.g., acover of the lyophilization nest) so as to allow the exchange of airbetween the interior and exterior of the lyophilization nest. Forexample, the lyophilization nest may comprise a base and a coverconfigured to collectively define a nest interior space. Thelyophilization nest may comprise a gasket situated between the base andthe cover to form an air-tight seal therebetween when the base and thecover are in a closed relationship. The base may define a bottom plateconfigured to support one or more receptacles within the nest interiorspace, and the cover may define the one or more vent holes through a topplate thereof. The vents may have corresponding sealing elements incloseable engagement with the vents configured to move between an openconfiguration and a closed configuration. Preferably, the sealingelements are configured as stopper-like plugs that are in slidingengagement with a vent hole, and slide between an open configuration anda closed configuration. When the vents holes of the lyophilization nestare closed, the lyophilization nest is isolated against theenvironmental factors exterior the lyophilization nest, thus impedingthe exchange of air between the interior and exterior of thelyophilization nest. The vent holes can be closed by a mechanismoperable from outside a sealed lyophilizer. For example, the sealingelements may be depressed into the closed configuration by lowering ashelf within the lyophilizer against the exterior surface of thelyophilization nest cover to thereby engage and depress the sealingelements into their respective vent holes.

The lyophilization nest containing the receptacle(s) is placed within alyophilizer with the one or more vent holes in the open position. Thelyophilization chamber is then sealed and substance(s) within thereceptacle wells are lyophilized. During the lyophilization process,atmospheric factors within the lyophilizer are altered to facilitateremoval of water from the substance(s). This controlled environmentwithin the lyophilization chamber consequently penetrates the interiorspace of the lyophilization nest through fluid communication with theone or more vent holes. The one or more vent holes are then closed,thereby sealing the lyophilization nest to impede rehydration of thesubstance(s) after completion of the lyophilization process bymaintaining within the lyophilization nest environmental conditions thatare favorable to maintaining the lyophilized substance(s). The seal onthe lyophilizer is later released and the lyophilization nest containingthe receptacle(s) is removed from the chamber. The lyophilization nestmay then be relocated and stored with the sample vessel positionedtherein until an operator is ready to use the lyophilized substance(s)located therein or to reseal the receptacle(s) containing thelyophilized substance(s) for further storage or for sale. Variousembodiments of the lyophilization nest may be utilized with alyophilizer having a vertically movable shelf that may be repositionedwhile the lyophilizer is sealed. Thus, the vertically movable shelf maybe lowered against the lyophilization nest to engage the sealingelements and to thereby seal the vent holes of the lyophilization nestwhile the controlled environment is maintained within the lyophilizer.

Various components of the lyophilization nest, the receptacle, the sealand similar components can be made using materials comprising a lowmoisture vapor transmission rate. Moisture vapor transmission rate is ameasure of the passage of water vapor through a substance. There are anumber of methods for determining the moisture vapor transmission rateof a material (e.g., numerous standard methods are described byInternational Organization for Standardization (ISO), American Societyfor Testing and Materials (ASTM), and others). In addition, materialswith reported moisture vapor transmission rate values are commerciallyavailable. One ordinarily skilled in the art will understand how tocalculate and/or purchase materials comprising a low moisture vaportransmission rate.

Lyophilization Nest

Referring now to FIG. 1, which shows an example lyophilization nest 1 ina closed configuration, the lyophilization nest 1 may define an airtight container configured to maintain a controlled environment therein.As shown in the embodiment of FIG. 1, the lyophilization nest 1 maycomprise a base 10 and a cover 50 having a gasket 60 situatedtherebetween to form an airtight seal between the base 10 and the cover50 when compressed between the base 10 and the cover 50. Referringbriefly to FIG. 2, the cover 50 defines one or more vent holes 53 withina top plate 52 configured to selectably place an interior space of thelyophilization nest 1 in fluid communication with an atmospheresurrounding the lyophilization nest 1. As shown in the illustratedembodiment of FIG. 1, each of the one or more vent holes 53 has acorresponding sealing element 100, such as a rubber plug or otherflexible plug. In the FIG. 1, each sealing element 100 is in slidableengagement with the corresponding vent hole 53 configured to engage aperimeter of the vent hole 53 and thereby form an air-tight sealtherein.

Moreover, in the illustrated embodiment of FIG. 1, the lyophilizationnest 1 includes one or more fasteners 150, embodied as latches,configured to selectably secure the base 10 and cover 50, and tocompress the gasket 60 between the base 10 and cover 50 to form anair-tight seal therebetween. However, a variety of fasteners may beutilized to secure the base 10 and cover 50 (e.g., clamps, screws,bolts, nuts, and the like). Moreover, in various embodiments the base 10and cover 50 may be secured via one or more hinges configured such thatthe base and cover may rotate around the hinge axis between an openrelationship and a closed relationship. In such configurations, one ormore fasteners may be utilized in conjunction with the one or morehinges such that the base 10 and cover 50 may be sealed in the closedrelationship to form an air-tight seal therebetween.

In various embodiments, a plurality of bases and/or a plurality ofcovers may collectively define the lyophilization nest 1. As anon-limiting example, two or more covers 50 may be configured to besecured in a closed configuration with a single base 10 to define alyophilization nest 1. Such plurality of covers may be configured toform an air-tight seal therebetween when each of the plurality of thecovers 50 is engaged in a closed configuration with the base 10. Forexample, one or more gaskets may be positioned between each of theplurality of covers to form an air-tight seal therebetween when thelyophilization nest is in the closed configuration. As an example, twocovers 50 may each be secured to opposing sides of a single base 10 viaone or more hinges. The two covers thus may be configured to rotatebetween an open configuration in which the covers do not impede accessinto the interior of the base 10, and a closed configuration in which anairtight seal is formed between the two covers and the base 10.

In various embodiments, the base 10 comprises a metal material (e.g.,aluminum), and the cover 50 comprises a plastic material (e.g., ahigh-density polyethylene material, a polyvinyl chloride material, orthe like), although other materials are also contemplated. For example,each of the base 10 and cover 50 may comprise an aluminum and/or aplastic material. Each of the base 10 and the cover 50 are a materialthat at least substantially impedes the migration of moisture into theinterior space of the lyophilization nest 1. Thus, for example, anymaterial having a low-moisture transmission rate may be utilized (e.g.,a cyclic olefin copolymer material; Topas Advanced Polymers, Inc.,Florence, Ky.; cat. no. 8007S-04). As another example, other materialsmay be utilized in such quantities so as to provide a wall thickness ofthe base 10 and/or cover 50 so as to impede vapor transmission throughthe base and cover between the interior and exterior of thelyophilization nest 1. As a non-limiting example, each of the base 10and/or the cover 50 may comprise a metallic material, a plasticmaterial, a composite material, or the like.

FIG. 2, which illustrates an exploded view of the various components ofthe lyophilization nest 1, provides additional details of each of thevarious components of the lyophilization nest 1. In the illustratedembodiment of FIG. 2, the base 10 comprises a raised base edge 11surrounding a bottom plate 12 which, collectively with a top surface ofthe bottom plate 12, defines a base interior 15. As shown in FIGS. 1 and2, the base 10 may be at least substantially rectangular, although thebase 10 may have any of a plurality of shapes (e.g., round, triangular,and the like). In various embodiments, the raised base edge 11 comprisesa smooth sealing surface configured to engage the gasket 60 (not shownin FIG. 2) so as to provide an air-tight seal therebetween when thegasket 60 is compressed between the raised base edge 11 and acorresponding cover edge, as discussed herein.

In the illustrated embodiment of FIG. 2, the base interior 15 isconfigured to support a plurality of individual receptacles 200 therein.As will be described in greater detail herein, each of the plurality ofreceptacles 200 may define one or more substance wells each configuredto hold a substance to be lyophilized. For example, each of thereceptacles 200 may be a single well receptacle or a multi-wellreceptacle. The base interior 15 may comprise one or more receptaclereceiving portions 16 therein. Each of the one or more receptaclereceiving portions 16 may be integrated into the base 10. In variousembodiments, the one or more receiving portions 16 may be secured to thebase 10. Like the base 10, the one or more receptacle receiving portions16 may comprise aluminum; although a plurality of alternative materialsare contemplated. In various embodiments, the one or more receptaclereceiving portions 16 may be configured to conduct heat away from theone or more receptacles 200 positioned therein and/or may be configuredto support the one or more receptacles 200 in an upright position.Accordingly, the one or more receptacle receiving portions 16 may beconfigured to support and/or conform to an exterior shape of at least abottom portion of the one or more receptacles 200 to be positionedtherein to maximize a surface contact area between the receptacles 200and the corresponding receptacle receiving portions 16. Moreover, theone or more receptacle receiving portions 16 may comprise a materialhaving a high heat transfer coefficient (e.g., aluminum), and may be insubstantial contact with the base 10 such that heat transfer between theone or more receptacle receiving portions 16 and the base 10 ismaximized. Referring again to the example lyophilization nest 1illustrated in FIG. 2, the one or more receptacle receiving portions 16are formed from a single piece of material with the base 10. In variousembodiments, the one or more receptacle receiving portions 16 may beremovably secured to the base 10.

In the illustrated embodiment of FIG. 2, the lyophilization nest 1additionally comprises a cover 50 having a shape corresponding to thebase 10. In the illustrated embodiment, the cover 50 has a rectangularshape corresponding to the base 10. However, alternative shapes arecontemplated (e.g., round, triangular, and the like).

As shown in FIG. 3, which illustrates a bottom view of a cover 50according to various embodiments, the cover 50 has a cover edge 51around the perimeter of a top plate 52 and extending away from the topplate 52 to define a cover interior 55. In the illustrated embodiment,the cover edge 51 comprises an at least substantially smooth surfaceconfigured to form an air-tight seal with the gasket 60 (not shown inFIG. 3) and the corresponding base edge 11 (FIG. 2) when the gasket iscompressed between the cover edge 51 and base edge 11. Moreover, invarious embodiments, the gasket 60 may be secured to one of the cover 50or the base 10 (FIG. 2) such that a sealing portion of the gasket 60 islocated between the base edge 11 and the cover edge 51.

Although the illustrated embodiments of FIGS. 1-3 show the base edge 11and cover edge 51 as comprising substantially flat surfaces configuredto compress a gasket 60 therebetween to provide an air-tight seal,various embodiments may comprise other configurations for providing anair-tight seal between various components of the lyophilization nest 1.As a non-limiting example, the base edge 11 and cover edge 51 may havean interlocking configuration such that the surfaces of the base edge 11and cover edge 51 may comprise corresponding features such that the baseedge 11 and the cover edge 51 may interlock when the lyophilization nest1 is in the closed configuration. For example, the base edge 11 andcover edge 51 may have a tongue-and-groove configuration, such that atleast one of the base edge 11 and the cover edge 51 may comprise a tabconfigured to engage a corresponding groove on the other to form a sealtherebetween. In various embodiments, a gasket may be provided betweenthe base edge 11 and cover edge 51 (e.g., within the groove and/or onthe tab).

As shown in FIGS. 2 and 3, the cover 50 may define one or more ventholes 53 extending through the top plate 52 from a top surface to aninterior surface of the top plate 52 such that the interior volume ofthe cover interior 55 is in fluid communication with air surrounding thelyophilization nest 1 through a vent hole 53. Fluid communicationthrough a vent hole 53 permits the exchange of air between the interiorspace of the lyophilization nest 1 and the exterior of thelyophilization nest 1. In various embodiments, each of the one or morevent holes 53 may correspond to an individual compartment within thelyophilization nest 1 that, when the cover 50 is secured with the base10, is isolated from the other individual compartment(s). However, invarious embodiments, each of the one or more vent holes 53 may eachplace a single interior space of the lyophilization nest in fluidcommunication with the air surrounding the lyophilization nest 1. Asshown in FIG. 2, each of the vent holes 53 may have a correspondingsealing element 100 (e.g., a flap, a rubber plug or other flexible plug)in slidable engagement with the corresponding vent hole. The sealingelements 100 are configured to engage the perimeter of the vent hole 53and thereby form an air-tight seal within the vent hole 53, whichisolates the interior space of the lyophilization nest 1 (collectivelydefined by the base interior 15 and the cover interior 55) from the airsurrounding the lyophilization nest 1 exterior (external to the base 10and the cover 50). As shown in FIG. 2, at least a portion of the sealingelement 100 may be positioned external to the sealable lyophilizationnest 1, and may be configured to be depressed at least partially intothe top plate 52 to form an air tight seal in the corresponding venthole 53. Moreover, as shown in FIGS. 1 and 2, the top plate 52 maydefine an indentation 54 surrounding each of the one or more vent holes53. In the illustrated embodiment of FIGS. 1 and 2, the indentation 54may have a uniform depth across the entire indentation 54 thus definingan indentation surface on the bottom of the indentation 54, and may beconfigured such that a portion of the sealing element 100 is placedagainst the indentation surface when sealed within the correspondingvent hole 53. Moreover, the indentation 54 may have a depth such that atop surface of the sealing element 100 is at least substantially alignedwith the exterior surface of the top plate 52 when sealed within thecorresponding vent hole 53.

Although the illustrated embodiment of FIGS. 1-3 show the one or morevent holes 53 as extending through a top plate 52 of a cover, variousembodiments of the lyophilization nest 1 have vent holes 53 extendingthrough other portions of the lyophilization nest 1. For example, theone or more vent holes 53 may extend through a side of thelyophilization nest 1, and/or through the base 10. In variousembodiments, a lyophilizer may comprise a lyophilization nest sealingsystem configured to engage the one or more sealing elements and movethe one or more sealing elements to the closed configuration. In variousembodiments, the lyophilization nest sealing system may be configured tobe operated while the lyophilizer is sealed.

FIGS. 4A-4C illustrate various positions of the sealing element 100operable between an open configuration (FIGS. 4A & 4B) and a closedconfiguration (FIG. 4C) with respect to a corresponding vent hole 53.FIGS. 4A-4C illustrate the optional indentation 54, in which is venthole 53. As shown in FIGS. 4A and 4B, each of the sealing elements 100may define a sealing cap 101, a body portion 102, and a vent slot 103configured such that the interior space of the lyophilization nest 1 mayremain in fluid communication with the air surrounding thelyophilization nest 1 through the one or more vent holes 53 with thecorresponding sealing element 100 placed loosely therein. In variousembodiments, the sealing cap 101 may define a top surface of the sealingelement 100. As shown in FIGS. 4A-4C, the sealing cap 101 is illustratedas a round element having a flat top surface and a flat bottom surface.The sealing cap 101 is secured to a body portion 102 of the sealingelement 100. As shown in FIGS. 4A-4B, a top portion of the body portion102 of the sealing element is secured to a bottom surface of the sealingcap 101. In various embodiments, the edges of the sealing cap 101 mayextend beyond the perimeter of the body portion 102, such that a portionof the bottom surface of the sealing cap 101 is configured to be insealing contact with the top surface of the top plate 52 when thesealing element 100 is inserted into a corresponding vent hole 53 in theclosed configuration. With reference to FIGS. 4A-4B, as a specificexample, the body portion 102 and sealing cap 101 of the sealing element100 may be concentric and circular in shape, wherein the diameter of thesealing cap 101 is larger than the diameter of the body portion 102.

Moreover, as shown in FIGS. 4A-4B, the sealing element 100 may have avent slot 103 extending through at least a portion of the body portion102. For example, as shown in FIGS. 4A and 4B, the vent slot 103 mayextend from a bottom portion of the body portion 102 toward a topportion of the body portion 102 and terminate between the bottom portionand the top portion of the body portion 102. In various embodiments, thevent slot 103 may extend across the entire diameter of the body portion102. Moreover, in various embodiments, the sealing element 100 maycomprise a single piece of flexible material. Thus, with the sealingelement 100 placed in an open configuration in which the sealing element100 is placed loosely within a corresponding vent hole 53 such that aportion of the vent slot 103 is located external to the top surface ofcover 50 as shown in FIG. 4B, the interior space of the lyophilizationnest 1 remains in fluid communication with the air surrounding theexterior of the lyophilization nest 1. Once the one or more sealingelements 100 are pressed into the corresponding vent holes 53 in aclosed configuration, (FIG. 4C) such that the bottom surface of thesealing cap 101 is in sealing contact with a top surface of the cover50, the interior space of the lyophilization nest 1 is sealed from theexterior of the lyophilization nest and the interior space of thelyophilization nest is no longer in fluid communication with theexterior of the lyophilization nest. In various embodiments, the one ormore sealing elements 100 may comprise a rubber material, however aplurality of resilient and/or flexible materials are contemplated(preferably resilient and/or flexible materials having a low vaportransmission rate). Moreover, the one or more sealing elements 100 maybe configured to be operated while the lyophilization nest 1 is locatedwithin a lyophilizer. For example, as will be described in greaterdetail herein, the one or more sealing elements 100 may be configured tobe depressed into a sealed position within the corresponding vent holes53 by moving a vertically movable shelf or other actuated elementcapable of an automated movement within the lyophilizer toward theexterior surface of the lyophilization nest 1 (e.g., a downward movementtoward the cover 50) to engage the one or more sealing elements 100 andthereby alter the sealing elements and the corresponding vent holes 53from the open configuration to the closed configuration.

FIG. 5 illustrates a cross-sectional view of a lyophilization nest 1 ina closed and sealed configuration between a top shelf 301 and a bottomshelf 302 within a chamber 401 of a lyophilizer. As shown in FIG. 5,when the cover 50 is positioned such that the cover edge 51 is alignedwith the base edge 11, the base interior 15 and cover interior 55 formthe interior space of the lyophilization nest 1. In the illustratedembodiment of FIGS. 1, 2, and 5, the lyophilization nest 1 additionallycomprises one or more fasteners 150 configured to secure the cover 50relative to the base 10, and thereby maintain an air-tight sealtherebetween. As exemplified in FIG. 2, the one or more fasteners 150each comprise a base pin 151, a cam lever 152 and a link arm 153, thougha number of fasteners can be used to secure the cover 50 to the base 10.The fasteners are configured to selectively compress the gasket 60between cover 50 and base 10 by pressing the cover 50 toward the base10.

Moreover, as shown in FIG. 5, the one or more receptacle receivingportions 16 comprise features for supporting and/or conforming to atleast a bottom end of the receptacle 200 to be received therein. In theillustrated example of FIG. 5, the receptacle receiving portions 16 eachcomprise a plurality of receptacle well receiving features having ashape conforming to the exterior shape of wells 201 formed within thereceptacle 200. For example, for receptacle wells 201 having ahemispherical shaped exterior, the receptacle well receiving featuresmay have a corresponding hemispherical shape. Such corresponding shapemay maximize the surface contact area of each of the one or more wells201 in contact with the receptacle receiving portions 16 so as tomaximize conductive heat transfer between a substance located withineach of the one or more wells 201 and the base 10.

In the illustrated embodiment of FIG. 2, the lyophilization nest 1 isadditionally configured to support one or more receptacle frames 250each having one or more receptacle support portions 251. As shown inFIG. 2, each of the one or more receptacle frames 250 are configured tosupport one or more receptacles 200 therein, and thereby facilitateplacement and removal of the one or more receptacles 200 within thelyophilization nest 1. As shown in the illustrated embodiment of FIG. 2,the one or more receptacle frames 250 may be configured to be situatedon a top surface of the bottom plate 12 and positioned around the one ormore receptacle receiving portions 16 and to extend above a top surfaceof the base edge 11 when positioned within the base 10. Accordingly, theperimeter of the one or more receptacle frames 250 may thereby provide apositioning guide for ensuring the cover 50 is appropriately positionedrelative to the base 10 such that an air-tight seal may be formedtherebetween. However, in various embodiments, at least one of the cover50 and/or the base 10 may comprise one or more extrusions extendingbeyond a surface of the corresponding edges 11 and 51 to provide apositioning member for positioning the cover 50 relative to the base 10.Moreover, in various embodiments each of the one or more receptacleframes 250 may comprise aluminum, although any of a variety of rigidmaterials may be utilized (e.g., plastic, other metals, composites, andthe like).

FIGS. 6A-6C illustrate a receptacle 200 that may be positioned within alyophilization nest 1 according to various embodiments. As shown inFIGS. 6A & 6C, each of the one or more receptacles 200 may define one ormore wells 201 configured to receive a substance to be lyophilized. Forexample, each of the one or more receptacles 200 may comprise a singlewell or multiple wells. Each of the one or more receptacles 200 maycomprise a low vapor transmission rate material configured to impedemigration of water vapor through the material (e.g., a cyclic olefincopolymer), although other materials may be used (e.g., polypropylene).Moreover, each of the one or more receptacles may have a sealable uppersurface 202 configured such that a seal (e.g., an adhesive low moisturevapor transmission material that secures to the sealable upper surface202 of the receptacle 200) may be applied thereto and sealed such thateach of the one or more wells are individually sealed and isolated froman uncontrolled environment external to the sealed receptacle and/orwell. For example, as shown in FIG. 6B, a seal 203 may be secured andsealed to the top of the receptacle 200 and around each of the one ormore wells 201 (not shown in this Figure). In a preferred embodiment theseal 203 is a low moisture vapor transmission film such as a laminatestructure comprising a layer of aluminum foil, though the seal can bemade from other materials. Each of the one or more wells 201 may beaccessed individually by piercing through the seal 203, by peeling theseal 203 off of the sealable upper surface 202 or by otherwise removingthe seal from above one of the wells 201 to gain access to that well.Removing the seal 203 (e.g., piercing) to gain access to the lyophilizedsubstance in the below well 201 puts that well in fluid communicationwith the air external the receptacle 200. Lyophilized substance within awell 201 wherein the seal 203 has been removed can then be purposelyrehydrated or otherwise manipulated according to a desired use for thereconstituted reagent. In a sealed multi-well receptacle 200,lyophilized substances in other of the one or more wells 201 wherein theabove seal 203 has not been pierced or removed can be stored for lateruse. As an additional example, each of the one or more wells 201 mayhave an associated sealing cap configured to be secured within the wellto provide an air-tight seal around the perimeter of the well.

As shown in FIG. 6C, which is a cross-section of a row of wells 201 of areceptacle 200 according to various embodiments, each of the pluralityof wells may have an at least substantially hemispherical shaped bottominterior surface. Moreover, as shown in FIG. 6C, the exterior of each ofthe plurality of wells 201 may have a corresponding at leastsubstantially hemispherical surface. As described herein, at least thebottom end of the exterior surface of each of the plurality of wells 201may be configured to conform with a receptacle well receiving feature ofa receptacle receiving portion 16 of a lyophilization nest 1. Aspreviously noted, such corresponding features may maximize surface areacontact between the well 201 and the receptacle receiving portion 16 soas to facilitate heat transfer away from the substance positioned withinthe well 201.

Substances that can be lyophilized by the apparatus and methods for itsuse include, for example, pure chemicals, chemical mixtures, biologicsamples, such as cells, cell extracts or tissues, biological agents,such as nucleic acids, enzymes, antibodies and other proteins, labels,such as fluorophores, and combinations thereof. Additional examples ofsubstances that may be lyophilized also include various reaction mixesfor performing specific reactions, such as for performing PolymeraseChain Reactions (PCR), transcription mediated amplification, nucleicacid or protein capture assays, and nucleic acid or proteinhybridization assays.

A multi-well receptacle means a contiguous vessel that can contain atleast two substances such that they can be stored and manipulated inparallel but separately. Standard formats for multi-well receptaclesinclude 6, 24, 96, 384 or 1536 wells. The volume of each well in anexample 96 well format is about 300-400 μL with a working volume ofabout 75-200 μL. Volumes generally vary inversely with the number ofwells, typically in a range between 1 nL and 10 mL for each well,although other sizes are also contemplated. Exemplary wells can haveflat bottoms, hemispherical shaped bottoms, or V-shaped bottoms, amongother shapes.

As used herein, an example lyophilizer comprises a sealable enclosuredefining a chamber 401 configured to support one or more sample vessels(e.g., a multi-well sample vessel) and/or one or more lyophilizationnests 1 therein. The lyophilizer is configured to generate and maintaina controlled environment having conditions necessary to lyophilize oneor more substance(s) located within the sample vessels within thechamber 401. Exemplary lyophilizers are configured to adjust the levelsof a number of factors of the atmosphere, such as; air pressure/vacuumlevel, temperature, moisture content, and gas content. Preferably,lyophilizers adjust the air pressure factor of the atmosphere bylowering the pressure within the chamber 401 thereby generating a vacuumwithin the chamber 401. As a non-limiting example, the pressure withinthe chamber 401 of the lyophilizer may be lowered to 730 mTorr or less,and more preferably to 65 mTorr or less, or raised to greater than 760mTorr. 401. Lyophilizers also, preferably, remove heat from the chamber401 to thereby lower the temperature within the chamber 401. Thelyophilizer is thereby configured to generate an atmospheric temperaturewithin the chamber 401 in which any water within a substance will freezeand, in conjunction with an air pressure, will sublimate out of thesubstance. Lyophilizers also preferably alter the amount of a gas in thechamber 401, such as by increasing the amount of nitrogen in the chamber401. For example, after the lyophilization process is complete (e.g.,after substantially all of the water is removed from the substance(s)),the chamber 401 may be filled with an inert replacement gas (e.g., puredry nitrogen gas) and thereby raise the pressure within the chamber 401to at least substantially atmospheric pressure. For example, pure drynitrogen gas may be introduced to the chamber 401 of the lyophilizerwhile maintaining the pressure within the chamber 401 below atmosphericpressure (e.g., 730 mTorr or less). However, in various embodiments, thelyophilizer may not introduce an inert gas into the chamber 401, thusmaintaining a vacuum having a pressure less than 730 mTorr, andpreferably less than 65 mTorr within the chamber 401 of the lyophilizer.In yet other embodiments, an inert gas may be introduced to the chamber401 of the lyophilizer to raise the pressure within the chamber 401 toatmospheric pressure (e.g., 760 mTorr) or above. The negligible humidityof the resulting controlled environment (e.g., via sublimation followedby introduction of an inert gas) facilitates lyophilization of asubstance(s) and impedes rehydration of the lyophilized substance(s)after lyophilization. Such negligible humidity level may becharacterized in that a further reduction in humidity would not offer asignificant added benefit in impeding rehydration of the one or morelyophilized substances. Depending on the substance to be lyophilized,the levels of various environmental factors are adjusted and controlledso to lyophilize the substance. Those skilled in the art oflyophilization will readily develop and implement a set of controlledenvironmental conditions to achieve lyophilization of a substance ofinterest.

Lyophilizers can be adapted to include one or more vertically movableshelves and/or other actuated elements that may be repositioned using auser control system while the chamber 401 is sealed. Such verticallymovable shelves and/or other actuated elements may be utilized to applya pressure to the one or more sealing elements and thereby depress thesealing elements into respective vent holes in the lyophilization nest.In various embodiments, a lyophilizer may comprise a lyophilization nestsealing system configured to engage the one or more sealing elements ofthe lyophilization nest and move the one or more sealing elements intothe closed configuration. For example, the lyophilization nest sealingsystem may comprise a sealing member configured to engage the one ormore sealing elements and move the sealing elements to the closedconfiguration. Collectively, the lyophilizer and the lyophilization nest1 define a lyophilization system.

Method of Use

With reference to FIGS. 7-9, which illustrate various steps for sealinga lyophilization nest 1, a method of using a lyophilization nest 1 tomaintain a controlled environment around one or more substances isdescribed herein.

According to various embodiments, one or more substances to belyophilized are placed within a receptacle 200. For example, each of aplurality of individual substances is placed within individual wells ofa multi-well receptacle 200. Although the receptacles 200 areillustrated as separate from the various lyophilization nest 1components, in various embodiments the one or more receptacles may beintegrated into one of the plurality of lyophilization nest components(e.g., the base 10). The one or more receptacles 200 are positioned on atop surface of the base plate 12 and within the base interior 15. Invarious embodiments, each of the one or more receptacles 200 arepositioned within the base interior 15 relative to a correspondingreceptacle receiving portion 16. For example, for a receptacle 200having one or more individual wells, the corresponding receptaclereceiving portions 16 may comprise individual well receiving portionshaving an interior surface corresponding to an exterior shape of each ofthe one or more individual wells of the receptacle 200. Moreover, invarious embodiments, each of the one or more receptacles 200 ispositioned within a receptacle frame 250 before being placed within thebase interior 15. For example, as shown in FIG. 2, four (4) individualreceptacles 200 may be positioned within a single receptacle frame 250which may then be placed in the interior of the base 15.

Once the one or more receptacles 200 having one or more substances to belyophilized are placed within the base interior 15, the cover 50 isplaced over the base 10 such that the base interior 15 and coverinterior 55 collectively form a nest interior space having the one ormore receptacles 200 positioned therein. In various embodiments, the oneor more fasteners 150 (e.g., latches, not shown) are engaged with thebase 10 and the cover 50 to compress the gasket 60 between the base edge11 and the cover edge 51, forming an air-tight seal therebetween whenthe base 10 and cover 50 are in the closed relationship. However, invarious embodiments, the cover 50 is placed loosely over the base 10such that the air-tight seal may be formed while the lyophilization nest1 is positioned within the chamber 401 of the lyophilizer.

As shown in FIG. 7, one or more sealing elements 100 (e.g., rubberplugs) are placed loosely within corresponding vent holes 53 in thecover 50 such that the interior space of the lyophilization nest 1remains in fluid communication with the environmental conditions aroundthe exterior of the lyophilization nest (e.g., conditions within achamber 401 of a lyophilizer) via the one or more vent holes 53. Asdescribed herein, with the one or more sealing elements 100 placedloosely within corresponding vent holes 53 such that the sealingelements 100 are in an open configuration and the interior space of thelyophilization nest 1 is in fluid communication with the exterior of thelyophilization nest 1 via the one or more vent holes 53, the sealingelements 100 are in slidable engagement with the corresponding ventholes 53 such that the sealing elements 100 may be depressed into theclosed configuration to seal the vent holes 53.

The assembled lyophilization nest 1 is then placed within a lyophilizerhaving a sealable enclosure defining a chamber 401 having a sealconfigured to maintain therein an environment for lyophilizing one ormore substances, and having at least one vertically movable shelfconfigured for an automated movement and operable from the exterior ofthe lyophilizer while the chamber 401 is sealed. For example, thelyophilization nest 1 is placed on a bottom shelf 302 within the chamber401 below at least one top shelf 301. A controlled environment is formedwithin the chamber 401. The interior space of the lyophilization nest 1is in fluid communication with the chamber 401 and thus each of the oneor more substances is in contact with the controlled environment and islyophilized. After the one or more substances are lyophilized, and whilethe controlled environment remains within the chamber 401, each of theone or more vent holes 53 is sealed by depressing the correspondingsealing elements 100 into the vent holes 53 of the top plate 52 of thecover 50. One or more of the factors of the controlled environmentwithin the chamber 401 is then maintained in the interior space of thelyophilization nest 1 while the chamber 401 is unsealed allowing theenvironment in the chamber to change to include environmental factorlevels that are unfavorable to the lyophilized substance(s). FIGS. 8-9illustrate one embodiment for sealing a lyophilization nest 1 in achamber 401 so that the controlled environment in the chamber 401 can bereplaced with ambient air while the controlled environment is maintainedwithin the interior of the sealed lyophilization nest 1.

As shown in FIGS. 8-9, a top shelf 301 within the chamber 401 is movedto engage the one or more sealing elements 100 and to contact theexterior surface of the top plate 52 of the cover 50 such that each ofthe sealing elements 100 are depressed into the corresponding vent holes53 to form an air-tight seal therein and thereby close the vent holes 53to seal the interior space of the lyophilization nest 1. As anon-limiting example, a vertically movable top shelf 301 may be loweredonto the exterior surface of the top plate 52 of the cover 50 while thesealing elements 100 are loosely positioned within the correspondingvent holes allowing for fluid communication from the lyophilizer chamberto the interior space of the lyophilization nest via exposed vent slot103 and vent hole 53 (FIGS. 7 & 8). Top shelf 301 vertically lowersfurther onto the exterior surface of the top plate 52 of the cover 50and fully depresses the sealing elements into the corresponding ventholes 53, as shown in FIG. 9. As another non-limiting example, a bottomshelf 302 having the lyophilization nest 1 positioned thereon may bemoved upward such that the exterior surface of the lyophilization nest 1contacts a surface located above the lyophilization nest 1, such thatthe sealing elements are depressed into the corresponding vent holes 53.As another non-limiting example, a lyophilization nest sealing systemmay engage the one or more sealing elements 100 and move the sealingelements into the closed configuration. Referring again to FIGS. 4B-4C,the sealing element 100 may be depressed into a corresponding vent hole53 from a loose position as shown in FIG. 4B to a sealed position asshown in FIG. 4C in which a portion of the sealing element 100 is incontact with a bottom indentation surface of a corresponding indentation54 and a top surface of the sealing element 100 is at leastsubstantially aligned with the exterior surface of the top plate 52.Particularly when sealing the one or more sealing elements 100 withinthe corresponding vent holes 53 while a vacuum is present within thechamber 401 of the lyophilizer, depressing the one or more sealingelements 100 until the top surface of the one or more sealing elements100 substantially aligns with the exterior surface of the top plate 52may prevent the one or more sealing elements 100 from becoming securedto a smooth surface of the top shelf 301.

In various embodiments, an air-tight seal may be formed between thecover 50 and the base 10 while the cover 50 and the base 10 are in aclosed relationship by depressing the cover 50 against the base 10 andthereby compressing the gasket 60 therebetween. As a non-limitingexample, when a vacuum is formed within the chamber 401 of thelyophilizer, the air-tight seal between the base 10 and the cover 50 maybe formed while the lyophilization nest 1 is positioned within thechamber 401. After the vacuum is released within the chamber 401,negative pressure within the interior space of the lyophilization nest 1may maintain the air-tight seal between the base 10 and the cover 50.

Once the interior space of the lyophilization nest 1 is isolated fromthe surrounding environment within the chamber 401 by an air-tight sealwithin each of the plurality of vent holes 53 and between the cover 50and the base 10, the environment within the chamber 401 of thelyophilizer can be replaced with air that is unfavorable tolyophilization (e.g., high moisture content) by allowing air exterior tothe lyophilization chamber 401 to flood into the chamber 401. Forexample, an access door and/or a vent of the chamber 401 may be openedand thereby the environment within the chamber 401 is replaced by theenvironment from the space external to the lyophilizer. Thus, thegaseous composition, air pressure, temperature and/or humidity levelwithin the chamber 401 may change to become equivalent to theenvironment surrounding the lyophilizer (e.g., the environment of theroom in which the lyophilizer is placed). Because the interior space ofthe lyophilization nest 1 is isolated from the surrounding environment,a controlled environment within the lyophilization nest 1 is maintained.

As a non-limiting example, a vacuum may be maintained within thelyophilization nest 1 for at least 4 hours. As another non-limitingexample, a flooded nitrogen environment may be maintained within thelyophilization nest 1 for at least 8 hours in various embodiments.Absolute humidity within the interior space of the lyophilization nest 1may remain at <0.23 g/m³ for at least four hours.

Lyophilized substance(s) in the lyophilization nest 1 are preferablysealed directly in the receptacle 200 for later use, which requires thatthe lyophilization chamber 401 is opened, the sealed lyophilization nest1 from therein is removed and located to a processing station, and thereceptacle(s) 200 sealed. From the time that the chamber 401 door isopened, during removal of the lyophilization nest 1 from the chamber 401to a processing station, and up until the time just prior to unsealingthe lyophilization nest 1 for access to the receptacle(s) 200 therein,the environmental conditions in the interior of the lyophilization nest1 are favorable to maintaining the lyophilized substance(s). To accessthe one or more receptacles 200 and the lyophilized substance(s) storedtherein, the cover 50 is removed from the base 10, and the receptacles200 removed from the lyophilization nest 1 and sealed for storage andlater use or otherwise additionally processed. Thus, the lyophilizedsubstance(s) within the lyophilization nest 1 have a reduced exposuretime to an uncontrolled environment, thereby impeding absorption ofwater and thereby maintaining the integrity of the lyophilizedsubstance(s).

When lyophilizing large batches of substances in a single lyophilizationtreatment, a plurality of lyophilization nests 1 are used to maintain acontrolled environment within the interior of each lyophilization nest 1and surrounding the lyophilized substances therein, as is describedherein. To accommodate a plurality of lyophilization nests 1 in achamber 401 the chamber may include three or more shelves. In aconfiguration wherein the chamber 401 includes three or more shelves,there is one true top shelf 301, one true bottom shelf 302, and at leastone middle shelf. In this configuration, each middle shelf can functionas both a top shelf 301 wherein it depresses a sealing element 100 onthe top surface of a below lyophilization nest 1, and a bottom shelf 302wherein a lyophilization nest 1 rests atop its top surface. Here, theplurality of lyophilization nests 1 sitting on a number of the three ormore shelves can be sealed in an accordion-like action between the threeor more shelves within the chamber 401. Other configurations are alsouseful for using a plurality of lyophilization nests 1 in a chamber 401.Once the plurality of lyophilization nests 1 are sealed, the lyophilizeris opened allowing air to flow into the chamber 401. A controlledenvironment is maintained in the interior space of the sealedlyophilization nests 1 while these lyophilization nests 1 aretransferred to a station for further processing of the one or morereceptacles 200 therein. A subset (e.g., 1 or less than all) of theplurality of sealed lyophilization nests 1 is then unsealed forprocessing of the receptacle(s) 200 therein. For example, one or more ofthe plurality of lyophilization nests 1 are unsealed by removing thelatch 150 and separating the cover 50 from the base 10. Receptacle(s)200 are covered with a seal 203 for storage and later use. An additionalsubset of the plurality of lyophilization nests 1 is then unsealed andsubsequently processed. In this example the plurality of sealedlyophilization nests 1 used for lyophilizing a large batch of substancesmaintained a controlled environment surrounding the lyophilizedsubstances in the interior space of each of the plurality oflyophilization nests 1 during the introduction of unfavorableenvironmental factors into the chamber 401 of the lyophilizer, duringtransportation of the plurality of sealed lyophilization nests 1 fromthe lyophilizer to the subsequent processing station, and while in thesuccession for subsequent processing.

As described, after releasing the seal on the lyophilization nest 1,lyophilized substance(s) can be used as is or the receptacle 200 can besealed for storage and later use of the one or more lyophilizedsubstances contained in its one or more wells 201. Resealing may notremove ambient air present in the one or more wells 201 of thereceptacle 200, but such process isolates the substance(s) fromprolonged contact with the unfavorable environmental factors, and thusgreatly improves storage of the lyophilized substance(s). A receptacle200 can be sealed by removing the receptacle from the lyophilizationnest 1, and by applying and adhering a seal 203 (e.g. a low moisturevapor transmission film, such as, a laminate structure comprising alayer of aluminum foil) over the open face of the one or more wells 201so as to form an air tight seal between each of the wells of thereceptacle and the external surrounding air. Receptacles 200 comprisingmore than one well are preferably sealed with a seal 203 so that eachwell is individually enclosed, and so that the seal on each well to bebroken independently without exposing the remaining wells to unfavorableenvironmental conditions.

CONCLUSION

Many modifications and other embodiments of the lyophilization nest setforth herein will come to mind to one skilled in the art to which theapparatus and the methods of its use pertain, in response to having thebenefit of the teachings presented in the foregoing descriptions and theassociated drawings. Therefore, it is to be understood that thelyophilization nest and methods for its use are not to be limited to thespecific embodiments disclosed and that modifications and otherembodiments are intended to be included within the scope of the appendedclaims. Although specific terms are employed herein, they are used in ageneric and descriptive sense only and not for purposes of limitation.

We claim:
 1. A lyophilization nest sealing system comprising: a sealable enclosure defining a chamber; a lyophilization nest contained with the chamber, the lyophilization nest comprising a base, a cover, and an interior space defined by the base and the cover, wherein the interior space contains a plurality of lyophilization wells, each well being configured to hold at least one substance to be lyophilized, and wherein the interior space of the lyophilization nest is in fluid communication with air external to the nest through one or more vent holes extending through at least one surface of the lyophilization nest; a sealing element associated with each of the one or more vent holes, and wherein each sealing element is selectively manipulable from an open configuration of non-sealing engagement with the associated vent hole to a closed configuration of sealing engagement with the associated vent hole; and an actuated element within the chamber capable of an automated movement to cause the sealing elements to engage with a contact surface to manipulate the sealing elements from their open configurations to their closed configurations, thereby closing the vent holes so that the interior space of the lyophilization nest is sealed off to air external to the nest and the absolute humidity within the interior space of the lyophilization nest is stabilized.
 2. The lyophilization nest sealing system of claim 1, wherein the sealing elements and associated vent holes are positioned on a top surface of the cover.
 3. The lyophilization nest sealing system of claim 2, wherein the sealable enclosure includes a bottom shelf within the chamber and the lyophilization nest is situated on the bottom shelf, and wherein the actuated element comprises a top shelf situated above the bottom shelf and configured for automated, downward movement toward the top surface of the cover of the lyophilization nest that causes the top shelf to engage the sealing elements, thereby manipulating the sealing elements from their open configuration to their closed configuration and closing the vent holes so that the interior space of the lyophilization nest is sealed off to air external to the nest and the absolute humidity within the interior space of the lyophilization nest is stabilized.
 4. The lyophilization nest sealing system of claim 2, wherein the actuated element comprises a bottom shelf on which the lyophilization nest is situated and the sealable enclosure includes a top shelf situated within the chamber above the bottom shelf, and wherein the bottom shelf is configured for automated, upward movement toward the top shelf that causes the top shelf to engage the sealing elements, thereby manipulating the sealing elements from their open configuration to their closed configuration and closing the vent holes so that the interior space of the lyophilization nest is sealed off to air external to the nest and the absolute humidity within the interior space of the lyophilization nest is stabilized.
 5. The lyophilization nest sealing system of claim 1, wherein the sealing elements and associated vent holes are positioned on a side surface of the lyophilization nest.
 6. The lyophilization nest sealing system of claim 1, wherein the sealing elements and associated vent holes are positioned on the base of the lyophilization nest.
 7. The lyophilization nest sealing system of claim 1, wherein: each of the sealing elements is a flexible plug that comprises a sealing cap and a body portion depending therefrom, the body portion being in closeable engagement with an associated one of the vent holes and having one or more vent slots formed therein; and each of the sealing elements is configured to be positioned in an associated one of the vent holes, such that (i) the sealing element is in the open configuration with respect to the associated vent hole when the interior space is in fluid communication with air external to the nest via the vent slots; and (ii) the sealing element is in the closed configuration with respect to the associated vent hole and the interior space is not in fluid communication with air external to the nest when a bottom surface of the sealing cap is in sealing contact with a top surface of the top plate.
 8. A method of sealing a lyophilization nest comprising a lyophilized substance, the method comprising the steps of: (a) positioning a lyophilization nest within a chamber of a sealable enclosure, the lyophilization nest comprising a base, a cover, and an interior space defined by the base and the cover, wherein the interior space contains a plurality of lyophilization wells holding at least one substance to be lyophilized, wherein the interior space of the lyophilization nest is in fluid communication with air external to the nest through one or more vent holes extending through at least one surface of the lyophilization nest, and wherein each of the vent holes has an associated sealing element in closeable engagement therewith; (b) closing the chamber containing the lyophilization nest; (c) creating lyophilization conditions within the chamber for a period of time sufficient for the substance contained in the lyophilization wells to be lyophilized; and (d) while the chamber is closed, automating the movement of an actuated element to cause the sealing elements to engage with a contact surface, thereby closing the associated vent holes so that the interior space of the lyophilization nest is sealed off to air external to the nest and the absolute humidity within the interior space of the lyophilization nest is stabilized.
 9. The method of claim 8, wherein the step of creating the lyophilization conditions comprises generating a vacuum and a temperature below freezing within the chamber, and wherein the absolute humidity within the interior space of the lyophilization nest is less than 2.3 grams of water per cubic meter of air after step (d).
 10. The method of claim 8, wherein the actuated element is a vertically movable shelf within the chamber configured to apply a pressure to the sealing elements and thereby depress the sealing elements into the associated vent holes.
 11. The method of claim 10, wherein the sealing elements and associated vent holes are positioned on a top surface of the cover, wherein the sealable enclosure includes a bottom shelf within the chamber, and the lyophilization nest is situated on the bottom shelf, and wherein the actuated element comprises a top shelf situated above the bottom shelf and configured for automated, downward movement, and wherein step (d) comprises moving the top shelf down until the top shelf engages the sealing elements, thereby manipulating the sealing elements from their open configuration to their closed configuration and closing the vent holes so that the interior space of the lyophilization nest is sealed off to air external to the nest and the absolute humidity within the interior space of the lyophilization nest is stabilized.
 12. The method of claim 10, wherein the sealing elements and associated vent holes are positioned on a top surface of the cover and wherein the actuated element comprises a bottom shelf on which the lyophilization nest is situated and the sealable enclosure includes a top shelf situated within the chamber above the bottom shelf and the bottom shelf is configured for automated, upward movement toward the top shelf, and wherein step (d) comprises moving the bottom shelf up until the top shelf to engage the sealing elements, thereby manipulating the sealing elements from their open configuration to their closed configuration and closing the vent holes so that the interior space of the lyophilization nest is sealed off to air external to the nest and the absolute humidity within the interior space of the lyophilization nest is stabilized. 